SEC columns must be well packed to ensure high-resolution separation. Because of the required column packing skill and large number of manual steps, SEC columns in general are more expensive than other chromatography columns are. Thus, it is worthwhile to keep them in good shape for a long time. Here Pia provides some tips and hints for protecting your SEC column investment.
There are many ways to maintain the high performance of a size exclusion chromatography (SEC) column and ensure a long lifetime. First, to avoid compressing the packed bed, it is very important to apply the recommended flow rate stated in the column instructions. Also, regular cleaning of the column will contribute to optimal performance. Below, I will give you some more tips to keep your column in good shape.
Protect your new SEC column from the start
Before connecting the column to your chromatography system, make sure there is no air in the tubing and valves ahead of the column. It is important to exclude air from entering the column when running SEC.
SEC columns from Cytiva are equilibrated with storage solution, which is degassed 20% ethanol. This storage solution needs to be washed out before using the column. Please ensure that you do not compress the column bed when doing this.
Because ethanol is a viscous solution, a slow flow rate must be used to avoid compressing the packed bed. I recommend using approximately 50% of the maximum flow rate for your column. You can find your column’s maximum flow rate in the product instructions. Some instructions also have recommended flow rates when using ethanol.
Follow your column’s performance over time
Following your SEC column’s performance over time can help you to avoid problems and troubleshoot them when they occur. It’s a good idea to perform an initial efficiency test to determine your column’s baseline of N/m (column efficiency) and As (peak symmetry):
See Appendix 1 in our Size exclusion chromatography handbook for more information.
Adapt the flow rate for your sample and temperature
In SEC the resin’s packed bed must be intact to give good separation, because the separation occurs in one column volume. Thus, it is important to prevent gap formation in the column. Reduce the flow rate 50% when you work in low temperature, such as in a cold room or a cold cabinet. Also, reduce the flow rate 50% whenever you are using viscous samples or buffers. So if you are working with viscous sample/buffers in a cold cabinet, use only 25% of your normal flow rate.
Clean your SEC column regularly
I highly recommend cleaning your column routinely, for example after each 10–20 runs. Cleaning removes any precipitated proteins or other contaminants that might have built up on the column. You should also clean the column if you see colored bands in the top of the column or if the backpressure has increased substantially.
SEC columns from Cytiva tolerate high pH well. They can be cleaned many times with NaOH. The concentration of NaOH depends on the resin type: use 0.5 M NaOH for Superose and Superdex columns but only 0.2 M NaOH for Sephacryl columns. Never store the columns in NaOH. Instead, equilibrate the column immediately after cleaning with two column volumes (CV) of water followed by two CV of running buffer.
Detailed cleaning protocols are found in the instructions for each size exclusion chromatography column.
Store your column in 20% ethanol
Whenever you are not using your SEC column for more than two days, I suggest storing it in 20% ethanol. Also, to prevent air from entering the column, connect the transport tool (provided with your new column) to the column outlet. Then, fill it with storage solution up to about 50% of its total volume.
Don’t forget to reduce your normal flow rate both when filling and when removing the ethanol.
If you follow these suggestions, your SEC column should perform well for a long time. For more tips, refer to our procedure, “Maintenance and cleaning of size exclusion chromatography columns”.